猪圆环病毒2b亚型Cap融合蛋白的原核表达及其免疫原性分析

目的原核表达猪圆环病毒2b亚型(porcine circovirus subtype 2b,PCV2b)Cap融合蛋白,并分析其免疫原性。方法以PCV2毒株(CAU0673)基因组为参考序列,His-Sumo-PCV2b-Cap-pUC57重组质粒为模板,设计特异性引物,PCR扩增目的片段;将其产物克隆至pMAL-C4x载体中,构建pC4x-PCV2b-Cap重组质粒,转化至大肠埃希菌BL21(DE3),IPTG诱导表达,表达产物进行直链淀粉树脂纯化。SDS-PAGE分析目的蛋白的可溶性,Western blot检测其反应原性;以纯化的融合蛋白免疫BALB/c小鼠,Western blot检测小鼠血清抗体特异性。结果经双酶切及测序鉴定,重组质粒pC4x-PCV2b-Cap构建正确;融合蛋白MBP-Cap(matlose binding protein Cap)最适诱导条件为1 mmol/L IPTG 37℃诱导6 h,其以可溶性形式存在,相对分子质量约为81000,与PCV2b阳性猪血清、兔抗MBPtag多克隆抗体及免疫小鼠抗血清均可发生特异性反应。结论成功构建了pC4x-PCV2b-Cap重组表达载体,获得了可溶性的MBP-Cap蛋白,其纯化蛋白具有较好的免疫原性。本文为PCV2b诊断试剂盒的研发奠定了理论基础。

Prokaryotic soluble expression and immunogenicity of Cap fusion protein of porcine circovirus subtype 2b

Objective To express the Cap fusion protein of porcine circovirus subtype 2b(PCV2b)in prokaryotic cells and analyze its immunogenicity.Methods Specific primers were designed using the genome of PCV2 strain in a soluble form(CAU0673)as reference sequence and recombinant plasmid His-Sumo-PCV2b-Cap-pUC57 as template,based on which PCV2b-Cap-pUC57 fragment was amplified by PCR and cloned into vector pMAL-C4x.The constructed recombinant plasmid pC4x-PCV2b-Cap was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified with amylose resin then analyzed for solubility by SDS-PAGE and for reactogenicity by Western blot.BALB/c mice were immunized with the purified fusion protein and determined for serum antibody specificity by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pC4x-PCV2b-Cap was constructed correctly.The optimal condition for expression of MBP-Cap(matlose binding protein Cap)fusion protein was induction with IPTG at a final concentration of 1 mmol/L at 37℃for 6 h.The expressed product,with a relative molecular mass of about 81000,existed in a soluble form and showed specific reactions with porcine sera positive for PCV2b,rabbit anti-MBP-tag polyclonal antibody and antisera of immu-nized mice.Conclusion The recombinant expression vector pC4x-PCV2b-Cap was successfully constructed,and soluble MBP-Cap protein with good immunogenicity was expressed,which provided a theoretical basis for the development of diagnostic kit of PCV2b.